Moisture barrier

ABSTRACT

1. IN A STRIP OF BIBULOUS MATERIAL, CAPABLE OF ABSORBING FLUID BY CAPILLARY ACTION, SAIS STRIP BEING DIVIDED INTO SEVERVAL ZONES WHICH MAY BE IMPREGNATED WITH VARIOUS MATERIALS THE IMPROVEMENT OF WHICH COMPRISES SAID STRIP HAVING AT LEAST ONE MOISTURE BARRIER ZONE AREA FORMED BY HAVING IMPREGNATED THEREIN A CHROME COMPLEX COORDINATION COMPOUND OF   R-C&lt;(-O-CR(-CL2)--&gt;O(-H)-CR(-CL2)--&gt;O=)   WHEREIN R REPRESENTS A C13 TO C18 FATTY ACID RADICAL AND HAVING FURTHER IMPREGNATED OVER AND EXTENDING OVER EITHER SIDE OF THE SAME AREA ETHYL METHACRYLATE-,ETHYL ACRYLATE COPOLYMER.

United States Patent 3,846,247 MOISTURE BARRIER Donald P. Kronish,Rockaway, Lee S. Zuritr", Parsippany,

William D. Young, Montclair, and Paul R. Lewandowski, Parsippany, N.J.,assignors to Warner-Lambert Company, Morris Plains, NJ. No Drawing.Filed Sept. 27, 1972, Ser. No. 292,701 Int. Cl. 'C12k l /00 US. Cl.195-127 3 Claims ABSTRACT OF THE DISCLOSURE The present inventionrelates to an improved moisture barrier which is particularly suited foruse in the manufacture of diagnostic strips used in the identificationof bacteria.

Diagnostic strips for the detection and identification of bacteria madeof bibulous materials on which are sup ported in various zones or areasubstrates and reagents are well known. Strips of this type aredescribed and claimed in US. Pats. Ser. Nos. 3,341,427 and 3,359,180granted to Evans et al.

Distinguishing characteristics of various species of bacteria aredetectable by use of a series of reagent impregnated strips.Identification of the Enterobacteriaceae is accomplished by the use of aseries of ten such strips. The identifying tests are directed to thedetection of the products of a specific enzyme activity of a particularorganism. The results of the test are interpreted using a combination ofa flow chart and checkerboard table.

Reagent impregnated test strips are not only convenient, easily handled,and more stable than so called classical tests, but they lend themselvesto obtaining results more rapidly with an accuracy equivalent to theclassical wet tests which were preferred previously. A further advantageis that the test reactions are completed in no more than four hours andtherefore do not require aseptic working conditions.

Test strips currently used for rapid identification of theEnterobacteriaceae include those which test for:

H S detection Lysine decarboxylase Voges-proskauer Malonate utilizationEsculin hydrolysis It is to be noted that several of these tests areuseful for purposes other than the rapid identification of theEuterobacteriaceae such as the simple determination of the presence ofnitrate radicals or sulfide ions.

A high grade bibulous paper such as Eaton-Dykeman No. 623 is the usualmaterial which is impregnated with the substrates and reagents requiredto produce the desired test strip. Other bibulous materials may be usedinstead of paper such as felt, porous ceramic strips, woven or matterglass fibers and the like.

There are certain considerations which obtain in most of the testprocedures. One is that the colonies of bacteria tested are usuallysuspended in an isotonic saline solution. This suspension is placed inan incubation tube in a rather small amount, usually in the order of 0.3ml. The suspension must contact the part of the strip bearing an initialsubstrate or reagent but must not be dissipated by capillarity. This isimportant since some reactions are read by color changes in the salinesolution, and other tests depend upon the reagent zones being contactedonly after incubation or upon contacting various zones in a specifiedsequence. Premature mixing of reagents could invalidate a test result.

The bibulous nature of the strip is necessary for impregnation to becarried out successfully. The same characteristic then requires that areliable moisture barrier be applied to one or more area to preventdissipation of the saline by absorption into the strip. This alsoinsures that reagents will not be prematurely contacted by the solutionor mixed in those tests in which the order in which they are used issignificant.

What can be done is incubate a suspension of unknown bacteria in salinewith a suitable substrate. The products of the bacterias enzymemetabolism accumulate in the saline after a period of time. The reagentor indicator zone is contacted by the incubated cell suspension bytilting the incubation tube. The result is determined by reading thecolor developed on the reagent zone.

The limiting of the inoculum containing saline to contact certain zonesfor predetermined periods has been accomplished by placing variousmoisture barriers on and in the bibulous strip. One such barrier wasformed by impregnating a chrome complex, hydrophobic material availableunder the trade name Quilon described in US. Pat. 3,341,427. Quilon is achrome complex coordination compound of the formula:

wherein R represents a C to C fatty acid radical.

Experience with this moisture barrier material has shown it to beextremely effective for this purpose. It suffers, however, from twosignificant drawbacks. This chrome complex produces a suflicientlyacidic environment to affect some of the more delicate tests and thepresence of chrome ions can prevent the bacterial growth which isnecessary to produce the metabolites which these tests detect.

A substitute barrier material was then utilized which was a colorlessethyl methacrylate and methyl acrylate copolymer in a toluene solventand known as Krylon 150 Crystal Clear. Krylon solution wasadvantageously modified by further diluting it with compatible thinnerssuch as toluene or alower aliphatic alcohol to improve its ease ofapplication. The range of such dilution when suitable is from 3 to 33%volume of added solvent per volume of acrylic resin solution. Where thetest strip is made from a suitable filter paper such as Eaton-Dykman No.623 the preferred combination on a volume/volume basis is Krylon and 15%ethyl alcohol U.S.P.

The barrier provided by Krylon alone was found to become impaired whenthe rolls of paper which have had the various zones impregnated withreagents applied by rollers Were cut to the final strip form by aconventional cutter. One run of several rolls was found to have producedpercentages of strips which had faulty moisture barriers ranging from 3%to greater than It is not clear how this failure occurred. It could havebeen a change in one of the variables over which control is difficult orimpractical. This could include the paper itself, the moisture contentof the paper, the coating solutions, the conditions or mechanics of theprocess.

The present invention relates to an improved moisture barrier whichstops the migration of various solutions up the test strip and whichcontinues to maintain its functional integrity after cutting. Themoisture barrier of the present invention comprises an initialimpregnation of the bibulous material with Quilon followed by a secondimpregnation over the first and extending over either side of it withKrylon. Experience with this moisture barrier has shown only negligibleleakers despite rigid examination of randomly sampled test strips forpurposes of quality control.

It must be taken into consideration that the preferred way of makingreagent impregnated test strips is by using a roll of suitable bibulouspaper as long as 300-400 feet and rolling on to it, lengthwise, atdiscrete intervals, the required substrates, reagents and moisturebarriers. This is usually accomplished by concurrent or successivepassing of the paper between rollers which pick up the impregnauts fromreservoirs and deposit them on the paper as it passes over them. Theamounts deposited are determined by several factors including thephysical characteristics of the impregnant such as viscosity,flowability and wettability, the width of the applicator wheel and thepressure of the applicator wheel on the paper.

It is obvious that more than one pass may be made by the paper throughthe impregnating process and that both sides of the paper may have thesame or different materials applied to the same zonal level. Themoisture barriers are applied on both sides, at the same level, toinsure complete penetration into the body of the paper.

The following example of providing a moisture barrier in accordance isgiven by way of illustration and not by way of limitation:

A 10 meter length of Eaton-Dykman No. 623 filter paper 83 millimeterswide was used as the bibulous material for impregnation. Twocompositions were prepared for combined use as a moisture barrier.

Quilon S, a chrome coordination complex which is made with stearic acidis supplied in a solution which is about 70% isopropyl alcohol and 30%of the chrome complex. The preferred dilution for the impregnant isprepared by taking 5 ml. of Quilon S as received, adding 20 ml.isopropyl alcohol and 50 ml. of distilled (deionized) water. Theapparent pH is adjusted to 3.5 using 0.05 Na'OI-I. This usually resultsin a final volume of about 100 ml. This solution is placed in areservoir from which it is fed to coat the surface of an applicatorwheel having a width of 5 mm. The clearance between the applicator wheeland the opposing surface is such that the rotating wheel both appliesthe impregnating solution to the paper and simultaneously advances thepaper. A preferred rate of advance is about 20-40 feet/min.

The second moisture barrier material is applied over the first andpreferably extending on either side thereof, and preferably so that thewidth of the area of the ethyl methacrylate and methyl acrylatecopolymer superim posed impregnation is about three times that of thechrome complex coordination compound area of impregnation and extendsabout equidistantly from it on either side. This is done by using a 15mm. wide applicator wheel centered over the same portion of the paper asthe first. The two moisture barrier compositions are completelycompatible. It is not necessary to wait for the first to dry beforeapplying the second.

The Krylon solution is supplied containing about 22 to 26% acrylic resinsolids in toluene and diluted with ethyl alcohol U.S.P. The impregnatingcomposition was prepared by placing 85 ml. of Krylon solution in agraduate cylinder and adding a sufljcie nt quantity of ethyl.

4 alcohol U.S.P. to bring the volume to ml. The solutions are completelymiscible and therefore require no special mixing. It was applied aspreviously described. Krylon drys at room temperature and low humidityin about 4-8 hours, and the Quilon S complex is cured in about one day.

The roll of impregnated test paper is then cut into strips 6.3 mm. wide.The strips thus produced were tested for moisture barrier integrity bypartially immersing the portion next to the barrier in isotonic salinesolution containing a dye. The integrity of the barrier is judged ashaving been maintained when the zone above the barrier remains free ofdye. None out of 200 samples tested failed after four days. The testswere not continued beyond this since the reagent impregnated test stripwould be exposed to the possibility of moisture barrier failure not morethan four hours:

What is claimed is:

1. In a strip of bibulous material, capable of absorbing fluid bycapillary action, said strip being divided into several zones which maybe impregnated with various materials, the improvement of whichcomprises said strip having at least one moisture barrier zone areaformed by having impregnated therein a chrome complex coordinationcompound of Cir- References Cited UNITED STATES PATENTS 3,341,427 9/1967Evans et al 103.5 R 3,359,180 12/1967 Evans et al 195103.5 R

ALVIN E. TANENHOLTZ, Primary Examiner R. J. WARDEN, Assistant ExaminerUS. Cl. X.R.

1. IN A STRIP OF BIBULOUS MATERIAL, CAPABLE OF ABSORBING FLUID BYCAPILLARY ACTION, SAIS STRIP BEING DIVIDED INTO SEVERVAL ZONES WHICH MAYBE IMPREGNATED WITH VARIOUS MATERIALS THE IMPROVEMENT OF WHICH COMPRISESSAID STRIP HAVING AT LEAST ONE MOISTURE BARRIER ZONE AREA FORMED BYHAVING IMPREGNATED THEREIN A CHROME COMPLEX COORDINATION COMPOUND OF